Osmophore Diversity in the Catasetum Cristatum Alliance (Orchidaceae: Catasetinae)
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LANKESTERIANA 16(3): 317—327. 2016. doi: http://dx.doi.org/10.15517/lank.v16i3.26649 OSMOPHORE DIVERSITY IN THE CATASETUM CRISTATUM ALLIANCE (ORCHIDACEAE: CATASETINAE) EVELYN P. FRANKEN1,2, LUDMILA M. PANSARIN1 & EMERSON R. PANSARIN1 1 Biology Department, Faculty of Philosophy, Sciences and Literature of Ribeirão Preto/University of São Paulo - FFCLRP/USP. Av. Bandeirantes, 3900. CEP 14040-901. Ribeirão Preto/SP. Brazil. 2 Author for correspondence: [email protected] ABSTRACT. Catasetum is a neotropical orchid genus of 130 species characterized by its unisexual flowers. They are pollinated by male Euglossini bees. It is widely know that these bees collect volatile compounds in Catasetum from structures called osmophores. However, there is little information on morpho-anatomy and histochemistry of secretory tissues for this neotropical genus and data are lacking. Based on these arguments members of the Catasetum cristatum alliance, namely C. arietinum, C. ariquemense, C. barbatum, C. carolinianum, C. cristatum, C. lanciferum, C. multifidum, C. multifissum, C. rivularium and C. semicirculatum were analyzed. The labellum of male flowers of this alliance is elongate and with fimbriate margins, possessing two protuberances and a median saccate portion. The labellum of female flowers is galeiform and there is no ornamentation. The entire adaxial surface of the labellum is secretory in both sexes, including the fimbriae of male flowers. The structure of the secretory tissue is similar among species, and they are composed of a simple epidermis and five layers of underlying parenchyma. In most species the epidermis is flat, and cells are elongated in the saccate portion. In C. ariquemense and C. carolinianum the epidermal cells are papillous, while in C. semicirculatum they have convex surface (male flowers). The histochemical analysis detected lipophilic droplets and starch grains inside the secretory cells. All characteristics observed are encountered in secretory tissues with high-energy demand, as is common in osmophores. KEY WORDS: Euglossini bees, floral glands, flower reward, pollination, volatile compounds Introduction. Nectar is the most common and 2009, Anton et al. 2012), Sievekingia (Curry et al. widespread floral reward among orchids (e.g., van der 1991), Kegeliella (Curry & Stern 1991), Cymbidium Pijl & Dodson 1966). However, a significant number of (Stpiczynska 1993), Gymnadenia (Stpiczynska 2001), species reward their pollinators (i.e., male euglossine Bulbophyllum (Teixeira et al. 2004, Nunes et al. 2014, bees) with volatile compounds. These volatile oils 2015, Kowalkowska et al. 2015), Ophrys (Ascensão et are produced in structures called osmophores (Vogel al. 2005, Francisco & Ascensão 2013), Cyrtopodium 1990). In Orchidaceae, two types of osmophores have (Pansarin et al. 2008), Cyclopogon (Wiemer et al. been reported, namely glandular and epidermal (e. 2009), Grobya (Pansarin et al. 2009), Acianthera (de g., Effmert et al. 2006). Osmophores are commonly Melo et al. 2010), Cycnoches (Anton et al. 2012), formed by a single layer of epidermal cells, with uni- Chlorea (Sanguinetti et al. 2012), Vanilla (Pansarin et or multicellular papillae, or several layers of cells. al. 2014), Cirrhaea (Pansarin et al. 2014), Gongora According to Vogel (1990), osmophores cells have big (Adachi et al. 2015) and Catasetum (Vogel 1963). nuclei, dense cytoplasm, large amount of starch and Catasetum Rich. (Orchidaceae) has approximately high number of mitochondria and other organelles. 130 species distributed mainly through the tropical The majority of these characteristics can be found in region of Central and South America (Romero et al. tissues with high metabolic activity, which is related 2009). The majority of the species occur in the Brazilian to the secretion process (Fahn 2000). Studies about Amazon, where this genus is well represented (Miranda osmophores anatomic structure has been conducted & Lacerda 1992). The species are indistinguishable in a number of unrelated orchid genera, such as in based on the morphology of their vegetative structures. Restrepia (Pridgeon & Stern 1983), Scaphosepalum The flowers are usually unisexual and, occasionally (Pridgeon & Stern 1985), Stanhopea (Stern et al. 1987, hermaphrodite (with no reproductive function). All three Curry et al. 1988, Curry et al. 1991, Pansarin & Amaral flower shapes (staminate, pistillate and hermaphrodite) Received 13 July 2016; accepted for publication 3 October 2016. First published online: 26 October 2016. Licensed under a Creative Commons Attribution-NonCommercial-No Derivs 3.0 Costa Rica License. 318 LANKESTERIANA have distinct morphology and may occur in the same position to the pollinarium adherence and deposition individual and sometimes in the same inflorescence (e.g., Dressler 1993). (Romero & Nelson 1986, Romero 1992, Gerlach Since the work of Vogel (1963), none further 2013). Within Catasetum some species alliances are information about flower anatomy of Catasetum recognized based on the morphology of male flowers and has been published. Additionally, no information particularly on the relative position of the staminodes on secretory tissues of species belonging to the (Bicalho & Barros 1988). The “C. cristatum alliance” Catasetum cristatum alliance is available. Based on belongs to the subsection Isoceras, which has parallel these arguments this present work aims to increase or symmetrical antennae (i.e., staminodes). The species the knowledge on flower anatomy of Catasetum, of this group exhibits a strong morphological similarity giving details on the structures related to the resource and new information are required for taxa delimitation production and the tissues involved in the fragrance (Lacerda 1998). secretion of selected species belonging to the “C. For more than fifty years is known that Catasetum cristatum alliance”. species are pollinated by male Euglossini bees (Hymenoptera, Apidae), which collect volatile Materials and methods. Ten species of the compounds at the flowers (Vogel 1963). In this case, Catasetum cristatum alliance were analyzed (Tab. 1). the fragrance compounds are volatilized and act as an The specimens were collected in natural areas from attractant and a resource for male euglossine bees that Brazil or bought from commercial orchids cultivators collect them with a hard hair structure present on their and maintained in the Orchidarium LBMBP of the anterior legs and are stored into the hind tibiae (Eltz et Department of Biology, FFCLRP, São Paulo University, al. 1999). These fragrances consist mainly on a variety in the city of Ribeirão Preto (approx. 21º10’S, 47º48’W; of terpenoids and aromatics that sometimes possess 546 m a.s.l.), state of São Paulo, southeastern Brazil. a species-specific composition (Hills et al. 1972, In some cases more than one individual of a same Gerlach & Schill 1991). During fragrance collection, locality were potted with Pinus bark in a single pot. bees incidentally contact the antennae of staminate The vouchers material (living plants) was deposited at (male) flowers and the pollinarium is deposited on the the orchidarium of the Molecular Biology and Plants bee’s body (mainly on the scutum). When visiting a Biosystematics Lab (Tab.1). pistillate (female) flower, the pollinia deposition in The morphological features of fresh flowers of the stigmatic cleft can occur (Dodson 1962, Dressler Catasetum cristatum alliance were examined under 1968). Flower morphology and osmophore disposition a binocular stereomicroscope. To determine the on the labellum may guide the pollinator for a correct osmophores location in each species, flowers were TABLE 1. Species analyzed, locality (states of Brazil), flower sex (Male/Female), number of specimens analyzed (N) and reference number (RN) at LBMBP Orchidarium. Species Provenance Flower sex N RN Catasetum arietinum Miranda & Lacerda PE M 1 844 Catasetum ariquemense Miranda & Lacerda RO M 1 924 Catasetum barbatum (Lindl.) Lindl. AM M 1 952 Catasetum carolinianum Miranda & Lacerda GO M 1 946 Catasetum cristatum Lindl. co* M 1 990 Catasetum lanciferum Lindl. MG M 3 943 Catasetum multifidum Miranda PA M and F 2 815, 818 Catasetum multifissum Senghas RO M 1 916 Catasetum rivularium Barb.Rodr. AM M 1 955 Catasetum semicirculatum Miranda RO M 1 917 * Commercial orchidary. LANKESTERIANA 16(3). 2016. © Universidad de Costa Rica, 2016. FRANKEN et al. — Osmophores in C. cristatum alliance 319 collected during the fragrance production (2 to 5 protuberances are composed by a single tooth, three days after flower opening in daylight period) from or numerous teeth. In some cases, the apical callus (C. each specimen. The flowers were immersed in 0.1% barbatum and C. cristatum) or both calli (C. rivularium) (w/v) aqueous neutral red for 1-24 h (Vogel 1962). split in a cluster of fimbriae. In C. multifidum and Additionally, they were longitudinally sectioned with C. multifissum the apical portion of the labellum is a razor blade and immersed in a Lugol 1% solution tridentate and branched. In the rest of the labellum there for 1-10 min to localize tissue containing starch. The is no evident ornamentation and the surface is smooth. histochemical tests were made on transversal section of In female flowers ofC. multifidum, the secretory surface the labella stained with Sudan IV (Pearse 1985) to detect is smooth and the secretory tissue is homogeneously total lipids, and Lugol 1% (Hinchman 1973) for starch distributed. grains. To characterize the anatomical structure, flowers The structure